Cancer is the second most common cause of death in the U.S. and accounts for one of every eight deaths worldwide. During 2010, the American Cancer Society estimated approximately 1,529,560 new cancer cases would be diagnosed in the U.S. alone, and an estimated 569,490 Americans would die from cancer. In 2008, an estimated 12.4 million new cancer cases were diagnosed, and 7.6 million people died from cancer worldwide. Although medical advances have improved cancer survival rates, there is a continuing need for new and more effective treatment.
Cancer is characterized by uncontrolled cell reproduction. The cell division cycle, which regulates the transition from quiescence to cell proliferation comprises four phases: G1, S phase (DNA synthesis), G2, and M phase (mitosis). Non-dividing cells rest in quiescent phase, G0. The cell division cycle also has several checkpoint mechanisms, which arrest the cell cycle and induce the transcription of genes that facilitate the repair of cell damage. Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions. The major cell cycle checkpoints include the DNA Damage Checkpoint, during phases G1 and G2, and the Spindle Assembly Checkpoint, during M phase. These checkpoints ensure that critical events such as DNA replication and chromosome segregation are completed in high fidelity.
Regulation of the cell cycle checkpoints is a critical determinant of the manner in which tumor cells respond to many chemotherapies and radiation. Many effective cancer therapies work by causing DNA damage; however, resistance to these agents remains a significant limitation in the treatment of cancer. One important mechanism leading to drug resistance is the activation of a checkpoint pathway that arrests the cell cycle to provide time for repair. Through this mechanism cell cycle progression is prevented, and immediate cell death of the damaged cell may be avoided.
The cell division cycle involves various protein kinases that are frequently overexpressed in cancer cells. Examples of such cell cycle kinases include (1) the G1/S phase kinases: the cyclin-dependent kinases (CDK2, CDK3, CDK4, CDK6, CDK7, and CDK9), and cell division cycle 7 kinase (CDC7); (2) the DNA damage checkpoint kinases: Ataxia-Telangiectasia Mutated kinase (ATM), ATM and Rad 3-related kinase (ATR), the checkpoint kinsases (CHK1 and CHK2), WEE1, and myelin transcription factor 1 (MYT1); and (3) the mitotic kinases: CDK1, NIMA-related kinase 2 (NEK2), polo like kinase 1 (PLK1), Aurora A kinase, Aurora B kinase, Aurora C kinase, the Budding Uninhibited by Benomyl kinases (BUB1, BUB1B—also known as BUBR1, and BUB3), and the kinetochore kinase TTK (also known as MPS1). (Curr. Med. Chem. (2007) 14, 969-985). Because of their important role in the cell division cycle, these cell cycle kinases have been explored as targets for cancer therapy.
The Aurora kinases, first identified in yeast (Ipl1), Xenopus (Eg2) and Drosophila (Aurora), are critical regulators of mitosis. (Embo J (1998) 17, 5627-5637; Genetics (1993) 135, 677-691; Cell (1995) 81, 95-105; J Cell Sci (1998) 111(Pt 5), 557-572). In humans, three isoforms of Aurora kinase exist, including Aurora A, Aurora B and Aurora C. Aurora A and Aurora B play critical roles in the normal progression of cells through mitosis, whereas Aurora C activity is largely restricted to meiotic cells. Aurora A and Aurora B are structurally closely related. Their catalytic domains lie in the C-terminus, where they differ in only a few amino acids. Greater diversity exists in their non-catalytic N-terminal domains. It is the sequence diversity in this region of Aurora A and Aurora B that dictates their interactions with distinct protein partners, allowing these kinases to have unique subcellular localizations and functions within mitotic cells.
Overexpression of Aurora B kinase has been reported in some cancers, and has been correlated to a worsened prognosis in some cancers. (Mol Cancer Ther (2007) 6, 1851-1857). Aurora B kinase localizes to the centromeres in preanaphase cells. There it plays a critical role in spindle bipolarity and the establishment and maintenance of the spindle assembly checkpoint. (J Cell Biol (2001) 153, 865-880; J Cell Biol (2003) 161, 267-280; J Cell Biol (2003) 161, 281-294; Curr Biol (2002) 12, 894-899). Cells lacking Aurora B kinase function demonstrate a loss of normal chromosome alignment during mitosis due to a fast-acting and potent override of the mitotic spindle checkpoint. During telophase, Aurora B kinase localizes to the spindle midzone and midbody, respectively. There, Aurora B kinase functions in cytokinesis. (J Cell Biol (2001) 152, 669-682; Genes Cells (2005) 10, 127-137). Inhibition of Aurora B kinase through the use of gene mutations, RNA interference or ATP competitive selective small molecule inhibitors leads to defects in the attachment of the spindle microtubules to kinetochores, chromosome segregation and formation of the cleavage furrow. (J Cell Biol (2001) 153, 865-880; J Cell Biol (2003) 161, 267-280; J Cell Biol (2001) 152, 669-682; Mol Biol Cell (2003) 14, 3325-3341; Curr Biol (2002) 12, 894-899; Genes Cells (2005) 10, 127-137). Aurora B kinase inhibition also prevents the proper formation of the spindle assembly checkpoint, causing cells to exit mitosis prematurely without a mitotic arrest and often without completing cytokinesis. (J Cell Biol (2003) 161, 267-280; J Cell Biol (2003) 161, 281-294). These cells enter the G1 portion of the cell cycle with double the amount of DNA, in a process known as endoreduplication. Reports in the literature suggest that this endoreduplication event is a prerequisite for the antiproliferative and antisurvival effects of Aurora B inhibition. This effect may be related to the phosphorylation of the Rb tumor suppressor protein by Aurora-B, which might contribute to the cell cycle arrest in the postmitotic G1 phase on unscheduled exit from mitosis. In agreement with this, it was found that endoreduplication, and thus apoptosis, after Aurora-B inhibition by ZM447439 is not dependent on p53. (Mol Cancer Ther (2009) 8(7), 2046-56).
Although Aurora B kinase and Aurora A kinase are both members of the Aurora kinase family, they have distinct roles during the process of mitotic division. In the course of normal mitotic cell division, cells organize bipolar spindles, with two radial arrays of microtubules each focused into a spindle pole at one end, and connected to chromosomes at the other end. In the instant before sister chromatids segregate into daughter cells, the chromosomes are arranged in a straight line (the ‘metaphase plate’). This process of organizing bipolar mitotic spindles with fully aligned chromosomes serves to ensure the integrity of a cell's chromosomal complement during mitosis.
The Aurora A gene (AURKA) localizes to chromosome 20q13.2 which is commonly amplified or overexpressed at a high incidence in a diverse array of tumor types. (Embo J(1998) 17, 3052-3065; Int J Cancer (2006) 118, 357-363; J Cell Biol (2003) 161, 267-280; Mol Cancer Ther (2007) 6, 1851-1857; J Natl Cancer Inst (2002) 94, 1320-1329). Increased Aurora A gene expression has been correlated to the etiology of cancer and to a worsened prognosis. (Int J Oncol (2004) 25, 1631-1639; Cancer Res (2007) 67, 10436-10444; Clin Cancer Res (2004) 10, 2065-2071; Clin Cancer Res (2007) 13, 4098-4104; Int J Cancer (2001) 92, 370-373; Br J Cancer (2001) 84, 824-831; J Natl Cancer Inst (2002) 94, 1320-1329). This concept has been supported in experimental models, demonstrating that Aurora A overexpression leads to oncogenic transformation. (Cancer Res (2002) 62, 4115-4122; Mol Cancer Res (2009) 7, 678-688; Oncogene (2006) 25, 7148-7158; Cell Res (2006) 16, 356-366; Oncogene (2008) 27, 4305-4314; Nat Genet (1998) 20, 189-193). Overexpression of Aurora A kinase is suspected to result in a stoichiometric imbalance between Aurora A and its regulatory partners, leading to chromosomal instability and subsequent transforming events. The potential oncogenic role of Aurora A has led to considerable interest in targeting this kinase for the treatment of cancer.
As a key regulator of mitosis, Aurora A plays an essential role in mitotic entry and normal progression of cells through mitosis. (Nat Rev Mol Cell Biol (2003) 4, 842-854; Curr Top Dev Biol (2000) 49, 331-42; Nat Rev Mol Cell Biol (2001) 2(1), 21-32). During a normal cell cycle, Aurora A kinase is first expressed in the G2 stage where it localizes to centrosomes and functions in centrosome maturation and separation as well as in the entry of cells into mitosis. In mitotic cells Aurora A kinase predominantly localizes to centrosomes and the proximal portion of incipient mitotic spindles. There it interacts with and phosphorylates a diverse set of proteins that collectively function in the formation of mitotic spindle poles and spindles, the attachment of spindles to sister chromatid at the kinetochores, the subsequent alignment and separation of chromosome, the spindle assembly checkpoint and cytokinesis. (J Cell Sci (2007) 120, 2987-2996; Trends Cell Biol (1999) 9, 454-459; Nat Rev Mol Cell Biol (2003) 4, 842-854; Trends Cell Biol (2005) 15, 241-250).
Although selective inhibition of Aurora A kinase results in a delayed mitotic entry (The Journal of biological chemistry (2003) 278, 51786-51795), cells commonly enter mitosis despite having inactive Aurora A kinase. Cells in which Aurora A kinase has been selectively inhibited demonstrate a variety of mitotic defects including abnormal mitotic spindles (monopolar or multipolar spindles) and defects in the process of chromosome alignment. With time, monopolar and multipolar spindles may resolve to form two opposing spindle poles, although some of these defects may lead immediately to cell death via defective mitoses. While spindle defects resulting from Aurora A kinase inhibition induce mitotic delays, presumably through activation of the spindle assembly checkpoint, cells ultimately divide at a frequency near that of untreated cells. (Mol Cell Biol (2007) 27(12), 4513-25; Cell Cycle (2008) 7(17), 2691-704.; Mol Cancer Ther (2009) 8(7), 2046-56.). This inappropriate cell division occurs following a slow-acting suppression of the spindle assembly checkpoint due to loss of Aurora A kinase function. (Cell Cycle (2009) 8(6), 876-88). Bipolar spindles that are formed in the absence of Aurora A kinase function frequently show chromosome alignment and segregation defects, including chromosome congression defects at metaphase, lagging chromosomes at anaphase, and telophase bridges. Consistent with the chromosome segregation defects, cells treated with MLN8054, a selective inhibitor of Aurora A kinase, develop aneuploidy that increases over time. Subsequent to repeated passages through defective mitotic divisions, cells treated with MLN8054 will often undergo senescence, an irreversible growth arrest with distinctive morphological characteristics. (Mol Cancer Res (2010) 8(3), 373-84). In some cell lines, MLN8054-treated cells exit from mitosis and activate a p53-dependent postmitotic G1 checkpoint, which subsequently induces p21 and Bax, leading to G1 arrest followed by the induction of apoptosis. (Mol. Cancer. Ther (2009) 8(7), 2046-56). Some cells may also exit mitosis without cytokinesis. These cells enter the G1 phase of the cell cycle with double the normal DNA content and are therefore referred to as G1 tetraploid cells. Lastly, some cells may divide, albeit with severe chromosome segregation defects (Mol Cell Biol (2007) 27(12), 4513-25). In the latter two outcomes, the abnormal mitotic divisions result in deleterious aneuploidy leading to cell death or arrest. Alternatively, it is possible that a portion of these cells may be resistant to these terminal outcomes and can reenter the cell cycle, as aneuploidy has been demonstrated to be both a suppressor and a promoter of tumor cell growth.
Other targets for cancer therapy include the mitogen-activated protein kinase (MAPK) cascades, which are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. Of the known MAPK signaling pathways, the RAF-MEK-ERK pathway mediates proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes that promote tumor growth, progression, and metastasis. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression.
Extracellular-signal-regulated kinase (ERK) is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/threonine kinases. Raf activates the MAP kinase ERK kinase (MEK)1/2 dual-specificity protein kinases, which then activate ERK1/2. Additionally, the Raf-MEK-ERK pathway is a key downstream effector of the Ras small GTPase. Ras is a key downstream effector of the epidermal growth factor receptor (EGFR). ERK activation also promotes upregulated expression of EGFR ligands, promoting an autocrine growth loop critical for tumor growth. Other signal transduction pathways, such as the PI3K/PTEN/Akt pathway, interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets.
The frequent mutational activation of this pathway in human cancers points to an important role for this pathway in human oncogenesis. Ras small GTPase, the most frequently mutated oncogene in human cancers, is mutationally activated and/or overexpressed in a wide variety of human cancers. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways which serve to regulate its activity. Raf and Erk are also frequently mutated in a number of different tumor types.
Constitutive action of MAPKs has been reported in >30% of primary tumor cell lines including cell lines derived from colon, lung, breast, pancreas, ovary, and kidney. (Oncogene (1999) 18, 813-822). Higher concentrations of active MAPK/ERK (pMAPK/pERK) have been detected in tumor tissue as compared to normal adjacent tissue. (J. Clin. Invest. (1997) 99, 1478-1483).
Inhibition of Ras/Raf/MEK activity has been shown to be accompanied by a cell cycle arrest at the G0-G1 boundary, as well as in some cases, apoptosis mediated by the downregulation of the Bcl2 antiapoptotic protein, both of which act to block cell proliferation. A number of biochemical markers have been associated with this arrest, including upregulation of p21Waf1, p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. (Cancer Res (2005) 65(11), 4870-80).
Given the importance of the protein kinases involved in driving the cell cycle, it would be beneficial if more effective treatment regimens, which target these kinases could be developed. In particular, combined treatment regimens could be helpful for patients suffering from cell proliferative disorders, and might potentially even decrease the rate of relapse or overcome the resistance to a particular anticancer agent sometimes seen in these patients.
There is thus a need for new cancer treatment regimens, including combination therapies.